Lipophilic metal complexes for necrosis and infarction imaging

ABSTRACT

This invention describes the use of metal complexes that have a plasma protein bond of at least 10% as imaging diagnostic agents for locating an infarction or a necrosis using lasting positive visualization.

This application claims the benefit of the filing date of U.S.Provisional Application Serial No. 60/060,977 filed Oct. 6, 1997.

The invention relates to the subject that is characterized in theclaims, i.e., the use of metal complexes that have a plasma protein bondof at least 10% as imaging diagnostic agents for locating an infarctionor a necrosis based on the persistent accumulation of substances in theinfarction or necrosis area.

Detection, location, and monitoring of necroses or infarctions is animportant area in medicine. Myocardial infarction does not immediatelyresult in irretrievable, non-functioning tissue; rather, it initiates adynamic process that extends over a prolonged period (weeks to months).The disease proceeds in about three phases, which overlap rather thanbeing distinctly separated from one another. The first phase, thedevelopment of myocardial infarction, comprises the 24 hours after theinfarction, in which the destruction progresses like a shock wave (wavefront phenomenon) from the subendocardium to the myocardium. The secondphase, the already existing infarction, comprises the stabilization ofthe area in which the formation of fibers (fibrosis) takes place as ahealing process. The third phase, the healed infarction, begins afterall destroyed tissue is replaced by fibrous scar tissue. During thisperiod, extensive restructuring takes place.

Up until now, no precise and reliable process has been known that wouldmake it possible to diagnose the current phase of a myocardialinfarction in a living patient. For evaluating a myocardial infarction,it is of decisive importance to know the extent of the portion of tissuethat is definitively lost in the infarction and at what point the losstook place since the type of treatment depends on this information.

Infarctions occur not only in the myocardium but also in other tissues,especially in the brain.

While infarction can be healed to a certain extent, in the case ofnecrosis, locally limited tissue death, only the harmful sequelae forthe rest of the organism can be prevented or at least mitigated.Necroses can develop in many ways: due to injuries, chemicals, oxygendeficits, or radiation. As with infarction, knowing the extent andnature of a necrosis is important for further medical treatment.

It is known that infarction and necrosis can be represented byantibodies that are directed against biomolecules that occurintracellularly and by porphyrins, metalloporphyrins and theirderivatives. Antibodies and porphyrins can be produced only at greatexpense, however, and are problematical in terms of handling andcompatibility in several respects.

It has now been shown that, surprisingly enough, metal complexes thathave a plasm protein bond of at least 10% are suitable as imagingdiagnostic agents for locating necroses that are produced by infarctionor caused in some other way. In this case, the basic advantage consistsof a persistent positive (bright) dyeing of necrotic areas with littleto no signal enhancement of the environs. Non-protein-bonded, otherwisecomparable complexes lead for only a short time to signal enhancement ofwell-perfused tissue, whereby underperfused—even vital—tissues remainunaffected. The blood supply to the tissues can also be detected usingT₂ or T₂-star (susceptibility) effects, but differentiates non-vitalfrom necrotic tissue. The plasma protein bond is, as is familiar to oneskilled in the art, determined by equilibrium dialysis.

Preferably suitable are metal complexes that have a plasma protein bondof at least 50%, especially preferably of at least 80%. The metalcomplexes according to the invention have a molecular weight of at least350 Da, and preferably at least 400 Da.

They have a T¹-relaxivity of at least 2.0 [s⁻¹mM⁻¹], measured at 37° C.and 20 MHz in plasma (see, e.g., Chem. Rev. 1987, 87, 901). Theirstability constant is at least 10¹⁵ (logK=15).

The metal complexes according to the invention are metal derivatives of,e.g., polyaminopolycarboxylic acids, polyaminopolyphosphonic acids,porphyrins, texaphyrins, sapphyrins, peptides and their derivatives, asthey are described in, e.g.,

U.S. Pat. No. 5,403,576 WO 94/27644 EP 452 392 EP 391 766 U.S. Pat. No.5,512,294 U.S. Pat. No. 5,536,491 WO 95/09848 U.S. Pat. No. 5,462,725 WO95/32741 EP 425571 U.S. Pat. No. 5,562,894 WO 95/32004 U.S. Pat. No.5,407,657 U.S. Pat. No. 5,370,860 U.S. Pat. No. 5,463,030 WO 94/10182 JP05186372 U.S. Pat. No. 5,277,895 WO 93/16375 EP 413405 DE 43 02 287 EP352218 DE 40 11 684 EP 405704 DE 38 34 704 EP 292689 WO 97/26017 EP230893 WO 95/28179 U.S. Pat. No. 5,318,771 WO 89/05802 U.S. Pat. No.5,422,096 U.S. Pat. No. 4,899,755 U.S. Pat. No. 5,527522 U.S. Pat. No.5,250,285 WO 93/03351 WO 91/03200 WO 96/23526 EP 0722739 WP 95/28392 EP165716 EP 540075 U.S. Pat. No. 5,480,990 WO 95/32192 WO 95/31219 U.S.Pat. No. 5,358,704 U.S. Pat. No. 5,466,438 WO 92/11232 WO 95/31444 WO95/15319 WO 95/09161 U.S. Pat. No. 5,453,264 JP 05186372 EP 661279 WO94/03593 WO 97/30734 WO 97/30733 DE 44 05 140 GB 8903023 U.S. Pat. No.4,880,008. U.S. Pat. No. 5,583,220

If the metal complexes according to the invention are used for NMRdiagnosis, the metal must be paramagnetic. This can be an element fromthe series of transition metals or lanthanides. Suitable ions includethose of the elements iron, manganese, gadolinium, and dysprosium.

If the metal complexes according to the invention are used forradiodiagnosis, the metal must be radioactive. This can be an isotopefrom the series of elements Tc, In, Rh, Ga, Sc, Bi, Y, Fe, Sm, Ho, Co,Cu, Gd, and Eu.

As suitable chelating agents, the following can be mentioned by way ofexample:

2-(4-Ethoxybenzyl)-3,6,9-tris(carboxymethyl)-3,6,9-triazaundecane-1,11-dicarboxylicacid (ligand of Eovist®, EP 405704

2-(4-benzyloxybenzyl)-3,6,9-tris(carboxymethyl)-3,6,9-triazaundecane-1,11-dicarboxylicacid, EP 405704

2-(4-butylbenzyl)-3,6,9-tris(carboxymethyl)-3,6,9-triazaundecane-1,11-dicarboxylicacid, WO 95/28179

2,5,8,11-tetrakis(carboxymethyl)-2,5,8,11-tetraazabicyclo[10,4,0]-hexadecane,U.S. Pat. No. 5,358,704

2,5,12,15-tetrakis(carboxymethyl)-2,5,12,15-tetraazatricyclo[10,4,0,0^(6,11)]-icosane,U.S. Pat. No. 5,358,704

10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,WO 97/26017

10-[2-hydroxy-4-aza-5-oxo-7-oxa-10,10,11,11,12,12,13,13,14,14,15,15,16,16,17,17,17,-heptadecafluoroheptadecyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,WO 97/26017

2-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecan-1-yl]-3-benzyloxypropionicacid, WO 89/05802

2-benzyloxymethyl-3,6,9-tris(carboxymethyl)-3,6,9-triazaundecane-1,11-dicarboxylicacid, EP 230893

DTPA-Lys-Asp-Asp-4-pentylbicyclo[2,2,2]-octane-1-carboxylic acid,Mallinckrodt MP-2269, Vancouver SMRM, April 1997

4-[hydroxymethyl-(4,4-diphenyl)cyclohexyloxy-phosphoric aciddiester]-3,6,9-carboxymethyl-3,6,9-triazaundecane-1,11-dicarboxylic acid(MS-325), WO 96/23526

4-[hydroxymethyl-(10-phenyl)-decyloxy-phosphoric aciddiester]-3,6,9-carboxymethyl-3,6,9-triazaundecane-1,11-dicarboxylic acid(MS-323, WO 96/23526)

N-(4-Decylphenylcarbamoylmethyl)-diethylenetriamine-N,N′,N″,N″-tetraceticacid, EP 603403

4,5-Diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-bis[2-[2-(2-methoxyethoxy]ethoxy]-13,20,25,26,27-pentaazapentacyclo[20.2.1.]^(3,6).18,11.0^(14,19)]heptacosa-3,5,8,10,12,14,16,18,20,22,24-undecane.U.S. Pat. No. 5,583,220.

The production of the pharmaceutical agents is carried out in a wayknown in the art by the corresponding complex compounds—optionally withthe addition of the additives that are commonly used in galenicals—beingsuspended or dissolved in an aqueous medium and then the suspension orsolution optionally being sterilized. Suitable additives are, forexample, physiologically harmless buffers (such as, for example,tromethamine), additives of complexing agents or weak complexes (suchas, for example,

diethylenetriaminepentaacetic acid or the Ca complexes that correspondto the metal complexes according to the invention) or—ifnecessary—electrolytes such as, for example, sodium chloride or—ifnecessary—antioxidants such as, for example, ascorbic acid.

If suspensions or solutions of the agents according to the invention inwater or in a physiological salt solution are desired for enteral orparenteral administration or for other purposes, they are mixed with oneor more adjuvant(s) that are commonly used in galenicals [for example,methyl cellulose, lactose, mannitol] and/or surfactant(s) [for example,lecithins, Tween®, Myrj®] and/or flavoring substances for tastecorrection [for example, ethereal oils].

In principle, it is also possible to produce the pharmaceutical agentswithout isolating the complexes. Special care must always be taken toperform chelation in such a way that the complexes according to theinvention are virtually free of uncomplexed metal ions that have a toxicaction.

This can be ensured with the aid of, for example, color indicators suchas xylenol orange by control titration during the production process.The invention therefore also relates to the process for the productionof complex compounds and their salts. As a final precaution, thereremains purification of the isolated complex.

The pharmaceutical agents preferably contain 0.1 μmol-1 mol/l of thecomplex and are generally dosed in amounts of 0.0001-5 mmol/kg. They areintended for enteral and parenteral administration. The complexcompounds are used

1. for NMR diagnosis in the form of complexes of them with the ions ofelements with atomic numbers 21-29, 42, 44 and 58-70;

2. for radiodiagnosis in the form of complexes of them with theradioisotopes of elements with atomic numbers 27, 29, 31, 32, 37-39, 43,49, 62, 64, 70, 75 and 77.

The agents meet the varied requirements for suitability as contrastmedia for nuclear spin tomography. They are thus extremely well suitedfor improving the image, obtained with the aid of the nuclear spintomograph, as regards its informational value after oral or parenteraladministration by increasing the signal intensity. They also show thegreat effectiveness that is necessary to burden the body with thesmallest possible amounts of foreign substances, and the goodcompatibility that is necessary to preserve the noninvasive nature ofthe studies.

The good water solubility and low osmolality of the agents make itpossible to produce highly concentrated solutions, i.e., to keep thevolume load on the circulation within reasonable bounds and to offsetthe dilution by bodily fluids. In addition, the agents have not onlyhigh stability in vitro, but also surprisingly high stability in vivo,so that release or exchange of the bonded ions—which are inherentlytoxic—in the complexes occurs only extremely slowly within the timeduring which the contrast media are completely eliminated.

In general, the agents for use as NMR diagnostic agents are dosed inamounts of 0.0001-5 mmol/kg, preferably 0.005-0.5 mmol/kg. Owing totheir advantageous radioactive properties and the good stability of thecomplex compounds contained therein, the agents are also suitable asradiodiagnostic agents. Details on such use and dosage are described in,e.g., “Radiotracers for Medical Applications,” CRC Press, Boca Raton,Fla.

In in-vivo administration of the agents, the latter can be administeredtogether with a suitable vehicle such as, for example, serum or aphysiological common salt solution or together with a protein such as,for example, human serum albumin. In this case, the dosage depends onthe type of cellular disruption, the metal ion used, and the type ofimaging method.

The agents are usually administered parenterally, preferably i.v. Theycan also be administered—as already discussed—intravascularly orinterstitially/intracutaneously.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a-c are MRI images of a rat with induced renal infarctions

FIGS. 2a-c are MRI images of a rat with induced renal infarctions

FIG. 3a is a graph of Contrast (infarction in Myocardium) vs time (0-180minutes after i.v. administration of contrast medium)

FIG. 3b is a graph of Percentage Enhancement vs time (0-180 minutesafter i.v. administration of contrast medium)

FIGS. 4a-d are four MRI images of a rat (Han. Wistar, Schering SPF,male, ≈300 g) with myocardial infarction that is induced—by occulsion ofthe left coronary artery—before and after administration of MS-325 (100μmol of Gd/kg of body weight). (MR technology: SE_SAT, EKG-triggered,T_(R): about 400 ms, T_(E): 10 ms, nt=4, Ma: 128*256, FOV: 7*7 cm, SD≈3mm, 1 layer, axial)

FIG. 4a is precontrast

FIG. 4b is 6.5 minutes p.i.

FIG. 4c is 60 minutes p.i.

FIG. 4d is 19 hours p.i.

The examples below are used to give a more detailed explanation of thesubject of the invention:

MRI Experiments on Animals with Induced Renal Infarctions

Enhancement in the MRI experiment was studied after one-time intravenousadministration of the substance Eovist® in animals with experimentallyinduced renal necroses or infarctions. Plasma protein bond: 10% (Europ.Workshop on Magn. Reson. in Medicine, Santiago de Compostela, Spain,Sep. 28-30, 1994).

The induction of the renal infarctions was carried out on anesthetized(Rompun®/Ketavet®, i.p.) rats (Han. Wistar, Schering SPF, about 200 g ofbody weight) by occlusion of a (caudal) branch of the left renal artery.The contrast medium was administered (dose: 300 or 500 μmol of Gd/kg ofbody weight) about 24 hours after the induction of infarction. Theanimals were studied before and up to 24 hours after contrast mediumadministration by MR-tomography (SISCO SIS 85, 2 tesla; SE sequence,T_(R): 400 ms, T_(E): 15 ms, nt=4, ni=128, FOV: 12-7 cm, SD≈3 mm, 1layer each axial or coronary).

After the MRI experiments were completed, the anesthetized animals weresacrificed by exsanguination (via the V. cava), and both kidneys wereprepared. To verify the infarction (size and position), the left(infarcted) kidney was removed and sliced into disks, and then NBT(“vital”) coloring was carried out.

Before the contrast medium was administered, no differentiation waspossible between vital and avital (infarcted) areas in the (left,treated) kidney (see FIGS. 1a, 2 a).

Immediately after substance administration, the nonperfused portion ofthe kidneys in each case was shown as a hypointense area (see FIGS. 1b,2 b). Starting at about 15-30 minutes p.i., the signal intensityincreased somewhat in the non-perfused area or the size of the delimited(low-signal) area decreased (→slow diffusion in the necrosis). In thelate phase (about 4-6 hours p.i.), a considerable signal increase(enhancement) in the necrotic area of the kidneys was noted in all ofthe animals studied (see FIGS. 1c, 2 c). The delineation of the necroticarea in the MRI experiment correlated very well with the results of thehistological “vital” coloring.

MRI Experiments on Animals with Induced Myocardial Infarctions

Necroselective enhancement was studied after one-time intravenousadministration of the substance MS-325 (WO 96/23526, Example 10, Gd-DTPAderivative) in animals with experimentally induced myocardialinfarctions in the MRI experiment. The induction of the myocardialinfarctions was carried out on anesthetized (Domitor®/Dormicum® i.m.)rats (Han. Wistar, Schering SPF, male, about 300 g of body weight, N=10)by occlusion of the left coronary artery. The contrast medium wasadministered (initial solution diluted with blood, dose 100 μmol/kg,i.v. bolus) 24 hours after the induction of infarction. The animals werestudied before and up to 3 hours (1, 5, 10, 15, 30, 45, 60, 75, 90, 105,120, 135, 150 and 180 minutes) p.i. (see FIG. 3a) continuously and 24hours after the contrast medium administration by MR tomography (SE,SAT, EKG-triggered, T_(R): about 400 ms, T_(E): 10 ms, nt=4, Ma:128*256, FOV: 7*7 cm, SD≈3 mm, 1 layer each axial) (see FIGS. 4a-d).

After the 24 hours p.i., the animals—in the MRT—were killed by anarcotic overdose, and an MRI experiment on “freshly-killed” animals (noartifacts of movement) was performed. To verify the infarction (size andposition), the heart was prepared, cut into disks and subjected tocoloring with NBT (nitro blue tetrazolinium chloride). Subjectiveevaluation of the enhancement and correlation with the colored tissuewere carried out. The signal intensities were standardized to a GD-DTPAsolution (0.25 mmol/l) and the percentage enhancement S1 post-S1 pre)/S1pre 100% and the contrast S1 inf/S1 myocard were calculated.

In the healthy myocardium (septum) and in the muscle, maximumenhancement was shown immediately after the administration of substancewith 100% or 60%. The signal intensity then dropped and reached a valueof between 10 and 20% after 150 minutes (see FIG. 3b).

In the infarcted area, however, the signal intensity increased within60-75 minutes to about 130% and then remained almost unchanged (up to180 minutes) (see FIG. 3b).

In contrast, it was possible to observe a negative contrast (S1 inf/S1myocard<1) within the first 10 to 15 minutes. Starting approximatelyfrom the 30th minute, it was possible to ascertain a positive contrast(S1 inf/S1 myocard>1) (see FIG. 3b).

After one day p.i., approximately the starting intensity (≈5-15%) wasagain reached in all tissues, and a contrasting of the myocardialinfarction (S1 inf/S1 myocard≈0.98) could no longer be detected.

MS-325 shows suitability as an infarction contrast medium.

What is claimed is:
 1. A method of locating a necrosis in a patientcomprising administering a metal complex having at least 10% bondingaffinity to plasma protein and imaging areas of possible necrosis at atime after said administration sufficient for the positive enhancementof the image of said necrosis with respect to the environs thereof,wherein said metal complex is not a porphyrin.
 2. A method according toclaim 1, wherein said complex has a protein bonding affinity of at least50%.
 3. A method according to claim 1, wherein said complex has aprotein bonding affinity of at least 80%.
 4. A method according to claim1, wherein said complex has a molecular weight that is greater than 350Da.
 5. A method according to claim 1, wherein said complex has astability constant of at least 10¹⁵ (logK=15).
 6. A method according toclaim 1, wherein said complex contains a paramagnetic metal effectivefor NMR imaging.
 7. A method according to claim 1, wherein said complexcontains a radioactive metal for radiodiagnostic imaging.
 8. A methodaccording to claim 6, wherein said complex contains, as a paramagneticmetal, iron, manganese, gadolinium, or dysprosium.
 9. A method accordingto claim 7, wherein said complex contains, as a radioactive metalisotope, Tc-99m, In, Rh, Ga, Sc, Bi, Y, Fe, Sm, Ho, Co, Cu, Gd, or Eu.10. A method of claim 1 wherein said image is taken at least about onehour after said administration.
 11. A method according to claim 1,wherein said complex has a relaxivity that is greater than 2.0 at 20 MHzand 37° C. in plasma.
 12. A method of claim 1 wherein the ligand of saidcomplex is2-(4-Ethoxybenzyl)-3,6,9-tris(carboxymethyl)-3,6,9-triazaundecane-1,11-dicarboxylicacid,2-(4-benzyloxybenzyl)-3,6,9-tris(carboxymethyl)-3,6,9-triazaundecane-1,11-dicarboxylicacid,2-(4-butylbenzyl)-3,6,9-tris(carboxymethyl)-3,6,9-triazaundecane-1,11-dicarboxylicacid,2,5,8,11-tetrakis(carboxymethyl)-2,5,8,11-tetraazabicyclo[10,4,0]-hexadecane,2,5,12,15-tetrakis(carboxymethyl)-2,5,12,15-tetraazatricyclo[10,4,0,0^(6,11)]-icosane,10-[1-methyl-2-oxo-3-aza-5-oxo-5-{4-perfluorooctylsulfonyl-piperazin-1-yl}-pentyl]1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-[2-hydroxy-4-aza-5-oxo-7-oxa-10,10,11,11,12,12,13,13,14,14,15,15,16,16,17,17,17,-heptadecafluoroheptadecyl]-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,2-[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)-cyclododecan-1-yl]-3-benzyloxypropionicacid,2-benzyloxymethyl-3,6,9-tris(carboxymethyl)-3,6,9-triazaundecane-1,11-dicarboxylicacid, DTPA-Lys-Asp-Asp-4-pentylbicyclo[2,2,2]-octane-1-carboxylic acid,4-[hydroxymethyl-(4,4-diphenyl)cyclohexyloxy-phosphoric aciddiester]-3,6,9-carboxymethyl-3,6,9-triazaundecane-1,11-dicarboxylicacid, 4-[hydroxymethyl-(10-phenyl)-decyloxy-phosphoric aciddiester]-3,6,9-carboxymethyl-3,6,9-triazaundecane-1,11-dicarboxylicacid,N-(4-decylphenylcarbamoylmethyl)-diethylenetriamine-N,N′,N″,N″-tetraaceticacid, or4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-bis[2-[2-(2-methoxyethoxy]ethoxy]-13,20,25,26,27-pentaazapentacyclo[20.2.1]^(3.6)18,11.0^(14,19)]heptacosa-3,5,8,10,12,14,16,18,20,22,24-undecaene.13. A method of claim 1 wherein said necrosis is an infarction.
 14. Amethod of claim 1 wherein said time after administration is around 24hours.